Diagnosis and prognosis of wound infection by measurement of a phospholipase A2 in wound fluid

ABSTRACT

The present invention relates to the diagnosis, prognosis and/or treatment of wound infection by testing wound fluid for the presence of a marker which is present in an amount which is indicative of infection. The marker may be high molecular weight phospholipase A 2  (cPLA 2 ) or a marker which is correlated with cPLA 2 .

CROSS-REFERENCE TO RELATED APPLICATIONS

This application, which is the national stage of internationalapplication PCT/GB2005/002262 that was filed Jun. 9, 2005, claimspriority to, and the benefit of, GB0413078.7that was filed Jun. 11, 2004and benefit of U.S. Provisional Application No. 60/625,830 that wasfiled Nov. 8, 2004.

All documents cited herein are incorporated by reference in theirentirety.

TECHNICAL FIELD

The present invention relates to the diagnosis and prognosis ofinflammatory conditions, in particular wound infection, by testing woundfluid for the presence of a marker which is present in an amount whichis indicative of an inflammatory condition. The marker may be highmolecular weight phospholipase A₂ (cPLA₂) or a marker indicative of thepresence of cPLA₂.

The present invention provides methods of diagnosis and prognosis,methods of treatment, diagnostic devices (e.g. biosensors) and kits foruse in such methods.

BACKGROUND ART

In mammals, injury triggers an organized complex cascade of cellular andbiochemical events that result in a healed wound. Wound healing is acomplex dynamic process that results in the restoration of anatomiccontinuity and function; an ideally healed wound is one that hasreturned to normal anatomic structure, function and appearance.

Chronically contaminated wounds all contain a tissue bacterial flora.These bacteria may be indigenous to the patient or might be exogenous tothe wound. Closure, or eventual healing of the wound is often based on aphysician⁷s ability to control the level of this bacterial flora.Infection of wounds by bacteria delays the healing process, sincebacteria compete for nutrients and oxygen with macrophages andfibroblasts, whose activity are essential for the healing of the wound.Infection results when bacteria achieve dominance over the systemic andlocal factors of host resistance. Infection is therefore a manifestationof a disturbed host/bacteria equilibrium in favour of the invadingbacteria. This elicits a systemic septic response, and also inhibits themultiple processes involved in wound healing. Lastly, infection canresult in a prolonged inflammatory phase and thus slow healing, or maycause further necrosis of the wound. The granulation phase of thehealing process will begin only after the infection has subsided.

In clinical practice, a diagnosis of infection is based on the presenceof local pain, heat, swelling, discharge and redness, although manyclinical indicators, such as inflammation and discharge, have a lowpredictive value of infection in wounds. Definitive diagnosis isachieved by microbiological analysis of wound samples. Tissue biopsyprovides the most accurate results, but this is an invasive procedurethat is difficult to achieve for the mass of specimens required. Woundswabbing is the most common wound sampling method used in the UKalthough its clinical value has been questioned. Furthermore,microbiological diagnosis of wound infection can take 48 to 72 hours,which allows time for infection to further develop iffirst-line/best-guess treatment is not employed immediately.

There therefore remains a need in the art for a method for the earlydiagnosis and prognosis of wound infection, and for devices and wounddressings for use in carrying out such methods.

cPLA₂ are enzymes that hydrolyse the sn-2 position of membraneglycerophospholipids to liberate arachidonic acid. The activity of theseenzymes is therefore important in the production of eicosanoids (e.g.prostaglandins and leukoriennes) and therefore associated withinflammation and host defence mechanisms. A detailed review can be foundin Kudo and Murakami, Prostaglandins and other Lipid Mediators (2002)68-69, 3-58.

There are essentially two groups of PLA₂: high molecular weight PLA₂(cytosolic PLA₂) and low molecular weight PLA₂ (soluble PLA₂). High Mol.Wt. cytosolic cPLA₂ is normally located intracellularly and as suchwould not be expected to be present in wound fluid. There are threeisoforms of this cPLA₂:α (110 kda), β (85 kDa) and γ (60 kDa) (See Kudoand Murakami, 2002). The low molecular weight soluble PLA₂'s (sPLA₂) areknown to act intracellularly and, in contrast to cPLA₂, we have notfound any evidence that the low molecular weight form (sPLA₂) iselevated in infected wound fluid.

WO03/101487 describes a method of assessing neural inflammatory diseasein an animal by determining the level of phospholipase A₂ protein(including cPLA₂) in tissue or body fluid such as blood, plasma andcerebrospinal fluid. It does not appear to teach or suggest theassociation of cPLA₂ with extracellular wound fluid or as a marker ofbacterial infection.

WO00/54052 describes an assay for detecting cPLA₂ in or on red bloodcells, in particular for the diagnosis of diseases in which dysfunctionof cell signalling systems involving highly unsaturated fatty acids areimplicated. This reference does not appear to teach or suggest thetesting of extracellular fluid for the presence of cPLA₂ to diagnoseinfection of a wound.

Funakoshi et al. in Pancreas (1991) vol. 6(5), pp 588-594 disclose thatserum PLA₂ levels are elevated in the sera of patients with pancreaticdiseases and that the measurement of serum PLA₂ is useful for diagnosisand monitoring of pancreatitis. There is no teaching or suggestion anyassociation of cPLA₂ with extracellular wound fluid or as a marker ofbacterial infection.

DISCLOSURE OF THE INVENTION

The present invention relates to a marker of inflammation/infection thatcould be used as a target molecule in a new diagnostic/prognostic assay(e.g. laboratory based or point of care) to identify an inflammatorycondition such as infection. The marker may be high molecular weightphospholipase A₂ (cPLA₂) or a marker which is correlated with cPLA₂.

Western blot analysis of wound fluid from infected and non-infectedpatients shows that cPLA₂ and cPLA₂ fragments (three bands are present)are observed in infected wound fluid (IV13) and no immunoreactivity tocPLA or cPLA₂ fragments was seen in the non-infected wound fluid (seeattached FIG. 1). Based on the specificity of the antibody and themolecular weight it is believed that the three bands observed correspondto cPLA₂α (110 kda), cPLA₂β (85 kDa) and cPLA₂γ (60 kDA).

cPLA₂ normally acts intracellularly and is not normally present in anwound environment. The observation that isoforms of this protein arepresent in infected but not in non-infected wound fluid is thereforenovel and surprising. It is believed that the data presented hereinrepresents the first demonstration that the cytosolic form of PLA₂ iseven present in wound fluid.

The finding that cPLA₂ is present in infected fluid means that thedifferentiation between infected and basal levels is significant. cPLA₂(and fragments thereof) could therefore be used as a marker ofinflammatory conditions such as infection and components for detectingor measuring the same could be incorporated in any type ofdiagnostic/prognostic kit (lab based (e.g. ELISA) or point of care (e.g.an antibody type kit similar to commercially available pregnancy kits).Further, cPLA₂ and cPLA₂ fragments may not only serve as markers ofwound infection but may be useful as general host derived markers ofinfection. Similarly, markers indicative of the presence of cPLA₂ may beused.

A first aspect of the invention provides a method of diagnosing orprognosing wound infection, the method comprising testing wound fluidfor the presence or level of cPLA₂ or a marker which is indicative ofthe presence or level of cPLA₂ in wound fluid.

A second aspect of the invention provides a diagnostic device selectedfrom the group consisting of wound dressings and biosensors for use indiagnosing or prognosing wound infection, comprising components of anassay system for testing wound fluid for the presence or level of cPLA₂or a marker which is indicative of the presence or level of cPLA₂ inwound fluid.

A further aspect of the invention provides a diagnostic or prognostickit for the diagnosis or prognosis of wound infection, the kitcomprising a wound dressing or biosensor according to the second aspectof the invention.

A further aspect of the invention relates to the use of components of anassay system for identifying in wound fluid the presence or level of:(i) cPLA₂; or (ii) a marker indicative of the presence or level of cPLA₂in wound fluid, in the manufacture of a biosensor, wound dressing,diagnostic or prognostic kit for diagnosing or prognosing woundinfection in a patient.

A further aspect of the invention relates to a system for use in thediagnosis and treatment of wounds comprising a diagnostic deviceaccording to the invention, and a wound dressing comprising at least oneantimicrobial agent for application to the wound when the measuredpresence or level of cPLA₂ or of a marker which is indicative of thepresence or level of cPLA₂ is indicative of wound infection.

A further aspect of the invention relates to a method for the treatmentof a mammalian wound comprising the steps of measuring the presence orlevel of cPLA₂ or a marker which is indicative of the presence or levelof cPLA₂, in a wound fluid collected from the wound, and applying anantimicrobial wound dressing to the wound selectively if the saidpresence or level is indicative of wound infection.

Whilst the invention has been described in relation to cPLA₂ it isenvisaged that other lipases may find utility as markers of infection.

Testing the Wound Fluid

The test on the wound fluid may be qualitative. Alternatively, aquantitative or semi-quantitative test for the marker (i.e. cPLA₂ or amarker indicative of the presence or level of cPLA₂) may be performed.Thus, in one embodiment the concentration of the marker is measured.

In one embodiment, the method is performed on wound fluid or serum whichhas been removed from body (e.g. as a clinical swab or as a fluidsample). In another embodiment, the method is performed on would fluidin situ. For example, the wound fluid may be tested by means of animplanted device or dressing attached to patient. The decision as towhich method is used will depend upon the type of wound in question.

Various methods may be used to detect or measure the concentration ofthe marker. Suitable methods include those utilizing chemical orenzyme-linked reactions, or immunological (e.g. ELISA, western blots),spectrophotometric, colorimetric, fluorimetric, or radioactive detectionbased techniques. In one embodiment a dip-stick type test is provided.Such a test could be used in the community and by the patient allowingeasier and earlier diagnosis/prognosis.

For example, in the case of surface-exposed wounds, a clinical swab,dressing, “dipstick” or other biosensor device may be applied directlyto the surface of the wound. The device should contain the components ofthe assay system for detecting the marker so that the assay reaction mayitself proceed in situ. The device can then be removed from the woundand the signal measured by the appropriate means. In many cases, aphysician may not actually require an accurate assessment of the preciseconcentration of the marker, but may just wish to know whether there isa sufficient concentration of the marker to warrant prophylactic orcurative action as necessary. In these cases, visible assessment of thedressing may be sufficient to allow identification of the specific areasof infection. Unnecessary treatment of healthy granulating tissue canthen be avoided.

A dressing that allows mapping of the infected areas of a wound will bepreferable in certain instances. Diagnostic wound mapping sheets thatcould be adapted to the methods of the present invention are describedin GB-A-2323166 (application no. GB 9705081.9), filed on 12th Mar. 1997,the entire content of which is hereby incorporated by reference.

Immobilization of reaction components onto a dipstick, wound mappingsheet or other solid or gel substrate offers the opportunity ofperforming a more quantitative measurement. For example, in the case ofa reaction linked to the generation of a colour the device may betransferred to a spectrometer. Suitable methods of analysis will beapparent to those of skill in the art.

Immobilization of the reaction components to a small biosensor devicewill also have the advantage that less of the components (such asantibody, enzyme and substrate) are needed. The device will thus be lessexpensive to manufacture than a dressing that needs to have a largesurface area in order to allow the mapping of a large wound area.

Methods for the incorporation of the components of the assay reactiononto a clinical dressing, “dipstick” sheet or other biosensor areroutine in the art. See for example Fägerstam and Karlsson (1994)Immunochemistry, 949-970.

Suitably, the methods, uses and/or devices, of the present inventioncomprise an immunological binding partner for the cPLA₂ or for themarker which is indicative of the presence or level of cPLA₂. Suitableimmunological binding partners include antibodies, including bothpolyclonal antibodies and monoclonal antibodies. Antibodies which may beemployed in the present invention include Polyclonal antibody againstc-terminal end of PLA2(abcam 9014); and monoclonal antibody to cPLA2-USBiological Cat non. P4074-04 and are available commercially. Theimmunological binding partner may be immobilized or bound to a solidsubstrate in a device as described herein.

The detectable signal produced by the device according to the presentinvention is observable or measurable by a physical, chemical, orbiological means known to those of skill in the art. A detectable signalmay be a change in emission or absorbance of electromagnetic waves at acertain wavelength, hybridization or enzymatic reaction. In preferredembodiments, detectable signals are changes in colour when viewed underwhite light or fluorescence when viewed under UV light. In certainembodiments, the device may comprise an electronic sensor, for exampleto detect color change or fluorescence and to provide a quantitativeoutput thereof The device may include an electronic sensor that canprovide a quantitative output in digital form.

The device may further comprise a reference assay element fordetermining the total protein content of the sample, so that themeasured level of cPLA₂ or of a marker which is indicative of thepresence or level of cPLA₂, can be normalised to constant total proteinlevel in order to increase accuracy.

In certain embodiments, the device according to the present inventioncomprises, or consists essentially of a wound dressing, dipstick orswab. In certain embodiments, the device according to the presentinvention comprises a housing containing one or more reagents and havingan inlet provided therein for introduction of the sample. The housingmay be at least partially transparent, or may have windows providedtherein, for observation of an indicator region that undergoes a coloror fluorescence change. In certain embodiments, the device operates onthe lateral flow principle. That is to say, said device comprises ahousing having an inlet for the sample and side walls defining a fluidlateral flow path extending from the inlet. By “lateral flow”, it ismeant liquid flow in which the dissolved or dispersed components of thesample are carried, preferably at substantially equal rates, and withrelatively unimpaired flow, laterally through the carrier. Suitably, thefluid flow path contains one or more porous carrier materials. Theporous carrier materials are preferably in fluid communication alongsubstantially the whole fluid flow path so as to assist transfer offluid along the path by capillary action. Suitably, the porous carriermaterials are hydrophilic, but preferably they do not themselves absorbwater. The porous carrier materials may function as solid substrates forattachment of reagents or indicator moieties In certain embodiments ofthe present invention, the device further comprises a control moietylocated in a control zone in said in said device, wherein the controlmoiety can interact with a component of the wound fluid sample toimprove the accuracy of the device.

The size and shape of the carrier are not critical and may vary. Thecarrier defines a lateral flow path. Suitably, the porous carrier is inthe form of one or more elongate strips or columns. In certainembodiments, the porous carrier is one or more elongate strips of sheetmaterial, or a plurality of sheets making up in combination an elongatestrip. One or more reaction zones and detection zones would thennormally be spaced apart along the long axis of the strip. However, insome embodiments the porous carrier could, for example be in other sheetforms, such as a disk. In these cases the reaction zones and detectionzones would normally be arranged concentrically around the center of thesheet, with a sample application zone in the center of the sheet. In yetother embodiments, the carrier is formed of carrier beads, for examplebeads made from any of the materials described above. The beads maysuitably be sized from about 1 micrometer to about 1 mm. The beads maybe packed into the flow path inside the housing, or may be captured orsupported on a suitable porous substrate such as a glass fiber pad.

It will be appreciated that the devices according to the presentinvention may be adapted to detect at least one analyte in addition tothe cPLA₂ or the marker which is indicative of the presence or level ofcPLA₂. This can be done by the use of several different reagents in asingle reaction zone, or preferably by the provision in a single deviceof a plurality of lateral flow paths each adapted for detecting adifferent analyte. In certain embodiments, the plurality of lateral flowpaths are defined as separate fluid flow paths in the housing, forexample the plurality of lateral flow paths may be radially distributedaround a sample receiving port. In some embodiments, the plurality offluid flow paths are physically separated by the housing. In otherembodiments multiple lateral flow paths (lanes) can be defined in asingle lateral flow membrane by depositing lines of wax or similarhydrophobic material between the lanes.

The devices according to the present invention may for example beincorporated into a bacterial sensing device of the kind described incopending application GB 0501818.9 filed on 28th Jan. 2005, the entirecontent of which is incorporated herein by reference.

Briefly, the devices of GB 0501818.9 are lateral flow sensors for thedetection of endogenous and/or microbial protease enzymes in wound fluidin order to ascertain the amount and type of bacterial infection. Thedevices comprise: a housing having an inlet for the sample and sidewalls defining a fluid flow path extending from the inlet, an indicatormoiety that is bound to a solid substrate by means of a peptide linkermoiety that is cleavable by the analyte enzyme, the solid substratebeing located in a reaction zone of the fluid flow path; and a detectormoiety located in a detection zone downstream from the reaction zone inthe fluid flow path, wherein the detector moiety can interact with anindicator moiety that has been cleaved from the solid substrate toproduce a detectable change in the detection zone.

An absorbent element may suitably be included in the devices of thepresent invention. The absorbent element is a means for drawing thewhole sample through the device by capillary attraction. Generally, theabsorbent element will consist of a hydrophilic absorbent material suchas a woven or nonwoven textile material, a filter paper or a glass fiberfilter

The device may further comprise at least one filtration element toremove impurities from the sample before the sample undergoes analysis.The filtration device may for example comprise a microporous filtrationsheet for removal of cells and other particulate debris from the sample.The filtration device is typically provided upstream of the sampleapplication zone of the fluid flow path, for example in the inlet of thehousing or in the housing upstream of the inlet.

Preferably, the devices according to the present invention include acontrol moiety in a control zone of the device, wherein the controlmoiety can interact with a component of the wound fluid sample toimprove the accuracy of the device. Suitably, the control zone isadapted to reduce false positive or false negative results. A falsenegative result could arise for various reasons, including (1) thesample is too dilute, or (2) the sample was too small to start with.

In order to address false negative mechanism , the control zonepreferably further comprises a reference assay element for determiningthe total protease content or the total protein content of the sample,that is to say for establishing that the total protease content or thetotal protein content of the sample is higher than a predeterminedminimum. It is possible to indicate the presence of protein by the useof tetrabromophenol blue, which changes from colorless to blue dependingon the concentration of protein present. It is also possible to detectglucose (using glucose oxidase), blood (using diisopropyl-benzenedihydro peroxide and tetramethylbenzidine), leukocytes (using ester anddiazonium salt). These may all be useful analytes for detection in thecontrol zone for the reduction of false negatives.

In a further aspect, the present invention provides a diagnostic testsystem or kit comprising a diagnostic device according to the presentinvention. The test system or kit may comprise, in addition to adiagnostic device according to the present invention, one or morecomponents selected from: a color chart for interpreting the output ofthe diagnostic device, a sampling device for collecting a sample of awound fluid from a wound, a wash liquid for carrying a sample of woundfluid through the device, and a pretreatment solution containing areagent for pretreatment of the wound fluid sample.

Where present, the sampling device may comprise a swab or a biopsypunch, for example a shaft having a swab or biopsy punch attachedthereto. Suitably, the diagnostic device includes a sample receivingport, and preferably the sample receiving port and the swab or biopsypunch comprise complementary fitting elements whereby the swab or biopsypunch can be secured to the device with the swab or biopsy punchreceived in the sample receiving port.

In certain embodiments the fitting element on the shaft may be locatedfrom 1 mm to about 30 mm from the base of the swab or the biopsy punch.This is consistent with the use of relatively small sample receivingport on the housing of the diagnostic device. The sample receiving portis typically located on an upper surface of the diagnostic device, andit is typically generally in the form of an upwardly projecting tube,open at the top and having the inlet to the fluid flow path located atthe bottom of the tube. Suitable swabs, biopsy punches and samplereceiving caps are described in detail in copending applicationsGB0403976.4 and GB0403978.0 both filed on 23rd Feb. 2004, the entirecontents of which are incorporated herein by reference.

The fitting element on the shaft may a tapered region of the shaft forforming an interference fit with the housing, for example it may appearas a truncated cone that is coaxial with the shaft and tapers towardsthe first end of the shaft. Or the whole shaft may have a diameterlarger than that of the swab or biopsy punch, with a tapered regionadjacent to the first end. In any case, the diameter of the taperedregion where it engages with the housing is normally greater than thediameter of the swab or biopsy punch, so that the inlet port can enclosethe swab or biopsy punch.

In other embodiments, the engagement element may comprise a snap-fittingprojection for forming a snap-fit with one or more complementaryprojections on an inner surface of the housing, or a threaded projectionfor forming a screw fit with one or more complementary threads on aninner surface of the cap, or a Luer-lock type fitting.

The swab may be any absorbent swab, for example a nonwoven fibrous swab.Typically the diameter of the swab is about 2 to about 5 mm, for exampleabout 3 mm. In certain embodiments, the swab may be formed from amedically acceptable open-celled foam, for example a polyurethane foam,since such foams have high absorbency and can readily be squeezed toexpel absorbed fluids. The biopsy punch will typically be a stainlesssteel cylindrical punch of diameter about 1 mm to about 10 mm, forexample about 3 mm to about 8 mm, suitably about 6 mm.

In certain embodiments the shaft is hollow, whereby a fluid can bepassed down the shaft from the second end to expel the biological samplefrom the swab or the biopsy punch into the diagnostic device. This helpsto ensure that all of the sample passes through the device, therebyavoiding false negatives. The shaft may comprise a fitting at the secondend for attachment of a syringe or other source of the fluid. In certainembodiments, the apparatus may comprise a reservoir of liquid attachedto the second end of the shaft, for example a compressible bulbcontaining the liquid, which can be activated after use of the swab orbiopsy punch. Suitable devices of this kind are described, for examplein U.S. Pat. No. 5,266,266, the entire content of which is incorporatedherein by reference. In other embodiments, the apparatus may comprise aplunger that can be pushed down the hollow bore of the shaft to expelfluid or other specimens from the swab or biopsy punch.

Another advantage of the hollow shaft is that, where the apparatus is abiopsy punch, the biopsy sample can more readily be pushed or blown outof the punch. The biopsy punch apparatus can further comprise ahomogenizing tool that can be passed down the hollow shaft to homogenizea tissue sample in the biopsy punch. This step of homogenizing can befollowed, if necessary, by passing liquid down the shaft from the secondend to expel the homogenized tissue from the biopsy punch into thedevice for diagnostic analysis.

In this aspect of the invention, the swab or biopsy punch may besterilized, and may be packaged in a microorganism-impermeablecontainer. The diagnostic devices according to the present invention mayalso be sterilized, but they may not, because the devices often do notcome into contact with the patient being diagnosed.

The concentration of the marker may also be measured in an aqueous assaysystem. For instance, wound fluid may be extracted directly from theenvironment of the wound or can be washed off the wound using a salinebuffer. The resulting solution can then be assayed for the concentrationof the marker in, for example, a test tube or in a microassay plate.

Such a method will be preferable for use in cases in which the wound istoo small or too inaccessible to allow access of a diagnostic/prognosticdevice such as a dipstick. This method has the additional advantage thatthe wound exudate sample may be diluted.

It will be clear that an aqueous assay system is more applicable to usein a laboratory environment, whereas a wound dressing containing thenecessary reaction components will be more suitable for use in ahospital or domestic environment.

The presence or level of a single or plurality (e.g. 2, 3, 4, 5 or more)of markers as defined in claim 1 may be assayed in the methods anddevices of the invention.

In a further aspect of the invention, there is provided a method ofdetermining whether a wound is infected, the method comprising testingwould fluid from the wound for the presence or level of cPLA₂ or amarker indicative of the presence or level of high molecular weightcPLA₂. Preferably, the method comprises the steps of contacting theindicator device of the invention with a sample, and assessing thegeneration of a detectable signal from the indicator device.

In certain embodiments according to this aspect, the method is adaptedto determine whether the presence or level of cPLA₂ or a markerindicative of the presence or level of high molecular weight cPLA₂ in asample of wound fluid exceeds a predetermined minimum level which ischaracteristic of an infection. In other embodiments, method is adaptedto determine whether the presence or level of cPLA₂ or a markerindicative of the presence or level of high molecular weight cPLA₂ in asample of wound fluid exhibits an increase over time that is indicativeof the development of infection.

In a further aspect, the present invention provides a method for thetreatment of a wound comprising the steps of measuring the presence orlevel of cPLA₂ or a marker indicative of the presence or level of highmolecular weight cPLA₂of a wound fluid collected from the wound, andapplying an antimicrobial wound dressing to the wound selectively if thesaid presence or level is indicative of wound infection. Preferably, themethod further comprises applying a wound dressing that is substantiallyfree of antimicrobial agents to the wound if the said presence or levelof cPLA₂ or a marker indicative of the presence or level of highmolecular weight cPLA₂ is indicative of absence of wound infection.

Preferably, the method according to this aspect comprises sampling thewound fluid at intervals, for example at intervals of from 1 hour to 24hours, and selecting an antimicrobial or non-antimicrobial dressing totreat the wound at said intervals in response to the presence or levelof cPLA₂ or a marker indicative of the presence or level of highmolecular weight cPLA₂. In certain embodiments, the wound dressingselection is determined by the cPLA2, whereby an antimicrobial dressingis applied when an increase in cPLA2 is detected, and a conventionalnon-antimicrobial dressing is applied if the cPLA2 is constant orfalling.

Preferably, the method her comprises determining the total proteincontent of the sample, whereby the presence or level of cPLA₂ or amarker indicative of the presence or level of high molecular weightcPLA₂ can be normalised to constant total protein content.

In a further aspect, the invention also provides a system for use in thediagnosis and treatment of wounds comprising a diagnostic deviceaccording to the invention and a wound dressing comprising at least oneantimicrobial agent The wound dressing comprising the antimicrobialagent(s) can be applied to the wound selectively, when the diagnosticdevice indicates the presence of wound infection.

Preferably, the system according to this aspect further comprises awound dressing that is substantially free from antimicrobial agents, forapplication to the wound when the measured presence or level of cPLA₂ ora marker indicative of the presence or level of high molecular weightcPLA₂ is indicative of a non-infected wound. The system may be in theform of a kit, and the device and the wound dressing(s) may be packagedtogether in a single package.

These aspects of the invention avoid unnecessary application ofantimicrobial agents to the wound, which is desirable because mostantimicrobial agents are cytotoxic and interfere with wound healing, andalso to avoid the development of resistant microorganisms.

The antimicrobial wound dressing used in these aspects of the inventioncomprises an effective amount of an antimicrobial agent, which maypreferably be selected from the group consisting of antiseptics andantibiotics and mixtures thereof Suitable antibiotics include peptideantimicrobials (e.g. defensins, Magainin, synthetic derivatives of them)tetracycline, penicillins, terramycins, erythromycin, bacitracin,neomycin, polymycin B, mupirocin, clindarnycin and mixtures thereof.Suitable antiseptics include silver sulfadiazine, chlorhexidine,povidone iodine, triclosan, other silver salts and colloidal silver,sucralfate, quaternary ammonium salts and mixtures thereof.

The wound dressing materials used in these aspects of the invention mayfor example be provided in the form of beads, flakes, powder, andpreferably in the form of a film, a fibrous pad, a web, a woven ornon-woven fabric, a freeze-dried sponge, a foam or combinations thereof.In certain embodiments, the dressing material is selected from the groupconsisting of woven fabrics, knitted fabrics, and nonwoven fabrics, allof which may be made by conventional methods. In other embodiments, thematerial may comprise (or consist essentially of) a freeze-dried spongeor a solvent-dried sponge

The wound dressing material may be in the form of a solid, or asemi-solid ointment or gel. Preferably, the wound dressing materialcomprises only up to 20% by weight, preferably less than 10% by weightof water. The relatively low water content improves the stability of thematerial and makes it possible to sterilize by heat or irradiationwithout loss of activity. The material may also contain 0-40% by weight,preferably 0-25% by weight of a plasticiser, preferably a polyhydricalcohol such as glycerol All of the above percentages are on a dryweight basis.

Any type of wound may be diagnosed or treated using the methods,apparatus and uses of the present invention. For example, the wound maybe an acute wound such as an acute traumatic laceration, perhapsresulting from an intentional operative incision. More usually the woundmay be a chronic wound. Preferably, the chronic wound is selected fromthe group consisting of venous ulcers, pressure sores, decubitis ulcers,diabetic ulcers and chronic ulcers of unknown aetiology. The presentinvention may be used in the diagnosis or prognosis of human andnon-human mammalian animals.

As used herein, the term wound fluid is meant to refer to the exudatethat is secreted or discharged by cells in the environment of the wound.The term “wound fluid” herein refers to any wound exudate or other fluidpreferably substantially not including blood) that is present at thesurface of the wound, or that is removed from the wound surface byaspiration, absorption or washing. The term “wound fluid” does notnormally refer to blood or tissue plasma remote from the wound site.

By testing wound fluid for the presence or level of cPLA₂, we includetesting for cPLA₂α, cPLA₂β or cPLA₂γ.

As an alternative to testing for cPLA₂ directly, a marker which isindicative of the presence or level of cPLA₂ may be detected ormeasured. For instance, binding proteins which specifically bind withcPLA₂ may be suitable markers of inflammatory conditions. Examplesinclude ERK1/2, P38 MAP kinase, S100 proteins, GTP binding proteins,annexins and calmodulin.

In addition as cPLA₂ acts on lipids other lipid binding proteins couldbe assayed such as prostaglandins, (e.g. PGE1) or fatty acid.

The skilled person will be able to verify the suitability of aparticular cytosolic PLA₂ binding protein or lipid binding protein as amarker of an inflammatory condition by experimentally verifying that thelevel of the marker in wound fluid (e.g. wound fluid) from healthypatients statistically differs from basal/normal levels of the markerand is thereby indicative of an inflammatory condition. Preferably, themarker is only present in negligible amounts (if at all) in the woundfluid of healthy patients.

The level of the marker is significantly different than the control(normal level) if the level of the marker differs from that of thecontrol level by an amount greater than the standard error of the assayemployed, and preferably at least twice, and more preferably three,four, five or ten times that amount. Alternately, the level of themarker can be considered “significantly” different than the controllevel of the marker if the marker level is at least about 1.5, two,three, four, or five times, higher or lower, respectively, than thecontrol level of the marker.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows the results of a Western blot for cPLA₂ in infected andnon-infected wound fluid. NV and IV fluid diluted 1:10.

MODES FOR CARRYING OUT THE INVENTION EXAMPLE 1

Collection and Treatment of Wound Fluid—Removal of Infected andNon-Infected Wound Fluid

All patients enrolled in the study had venous leg ulcers of at least 30days duration and a surface area of at least 1 cm². Patients werediagnosed as ‘non-infected’, normal appearance of wound, or ‘infected’based on a minimum of 4 clinical signs and symptoms indicative ofinfection. Patients were excluded from the study if exposed bone withpositive osteomyelitis was observed. Other exclusion criteria includedconcomitant conditions or treatments that may have interfered with woundhealing and a history of non-compliance that would make it unlikely thata patient would complete the study. Wound fluids were collected from thepatients following informed consent being given from all patients ortheir authorized representatives The protocol was approved by the EthicsCommittee at the participating study center prior to commencement of thestudy. The study was conducted in accordance with both the Declarationof Helsinki and Good Clinical Practice.

Sample Preparation

Total protein in each extracted wound fluid sample was determined usingthe Bradford protein assay, following manufacturers instructions for useof a Bio-Rad Protein Assay kit. All non-infected samples were then codedby NV followed by a specific number and the infected samples coded by IVfollowed by a number. Neat samples were diluted in PBS as 1:5 and 1:10and then mixed by pipetting with reduced treatment buffer—20 ul woundfluid mixed with 5 ul reduced treatment buffer and then boiled for 5mins in a water bath, to degrade proteins to fragments and producereduced samples. (NB tops of the eppendorf tubes must be open).Following this treatment all samples were frozen in −20° C. untilrequired for western blot analysis.

Western Blotting

Wound fluid from non-infected patient identified as NV12 and an infectedpatient IV13 was used. Broad range SDS page marker (Catalog number161-0318 obtained from Biorad) was used as a standard and 10% Tris-HClgels (Biorad) used. Samples were loaded in the gel and SDS pageelectrophoresis carried out at 60 mA until the samples ran to the bottomof the gel. Gels were transferred onto nitrocellulose paper of pore size0.45 um (Biorad) at 200 mA over 3 hrs whilst immersed in ice. Followinggel transfer, nitrocellulose paper was washed in PBS/0.1% Tween solutionfor 5 mins and then blocked in 5% milk for 1 hr at room temperature, RT,on shaker. Following another wash in PBS/Tween solution, one blot wastreated with primary antibody (abeam one polyclonal antibody, abeam9014) diluted in PBS/Tween solution and the second blot was treated withonly PBS/Tween solution as a control. Both blots were left on the shakerovernight at RT. The polyclonal primary antibody was obtained fromAutogen Bioclear UK (sc20105) at a concentration of 200 ug/ml anddiluted −100 ul neat added to 9,900 ul PBS/Tween solution. Followingincubation with the primary antibody, blots were washed every 5 mins inPBS/Tween solution for 30 mins and then treated to secondary antibody(anti-sheep HRP-linked antibody obtained from Sigma) for 1 hr at RT onthe shaker. The blots were then developed using the Opti4 CN kit(Biorad) for 5-10 mins and the bands observed.

Additional Data

FIG. 1 shows information from one infected and one non infected patient.To confirm this observation the wound fluid of five non-infected (NV2,5, 9, 12 and 14) and five infected patients (IV 1, 4, 10, 13 and 22) wasassessed using the method as described above.

These results demonstrated that no cPLA₂ was observed in the noninfected patients and cPLA₂ was present in 4 out of five infectedpatients. This additional experiment confirms that cPLA₂ is a marker ofwound infection.

It will be understood that the invention has been described by way ofexample only and modifications may be made whilst remaining within thescope and spirit of the invention.

The invention claimed is:
 1. A method of diagnosing or prognosing woundinfection, comprising testing a wound fluid for the presence or level ofhigh molecular weight phospholipase A₂ (cPLA₂) or of a marker which isindicative of the presence or level of cPLA₂, wherein the presence orlevel of said cPLA₂ or said marker is indicative of wound infection,wherein the wound is a chronic wound selected from the group consistingof a venous ulcer, a pressure sore, a decubitis ulcer, a diabetic ulcerand a chronic ulcer of unknown etiology.
 2. The method, according toclaim 1, wherein a concentration of cPLA₂ or the marker indicative ofthe presence or level of cPLA₂ is measured.
 3. The method according toclaim 1 wherein the method is performed on wound fluid in situ.
 4. Themethod according to claim 1 wherein the method is an in vitro methodthat is performed on a sample of wound fluid that has been removed froma body.
 5. A method for the treatment of a mammalian wound comprisingthe steps of measuring the presence or level of cPLA₂ or a marker whichis indicative of the presence or level of cPLA₂ of a wound fluidcollected from the wound, and applying an antimicrobial wound dressingto the wound selectively if the said presence or level is indicative ofwound infection.
 6. The method according to claim 5, further comprisingapplying a wound dressing that is substantially free of antimicrobialagents to the wound if the said presence or level is indicative ofabsence of wound infection.
 7. The method according to claim 5, whereinthe method comprises sampling the wound fluid at intervals, for exampleat intervals of from 1 hour to 24 hours, and selecting an antimicrobialor non-antimicrobial dressing to treat the wound at said intervals inresponse to the measured presence or level of cPLA₂ or a marker which isindicative of the presence or level of cPLA₂.
 8. The method according toclaim 7, wherein the antimicrobial wound dressing is applied to thewound if said level is increasing over time, and the non-antimicrobialdressing is applied to the wound if said level is decreasing over time.9. The method, according to claim 5, wherein wound fluid is tested forthe presence or level of cPLA₂α, cPLA₂ β or cPLA₂γ.
 10. The method,according to claim 5, wherein the marker which is indicative of thepresence or level of high molecular weight cPLA2 is ERK 1/2 , P38 MAPkinase, a S100 protein, a GTP binding protein, an annexin, calmodulin,PGE1 or fatty acid synthase.
 11. A method of diagnosing or prognosingwound infection, comprising testing a wound fluid for the presence orlevel of high molecular weight phospholipase A₂ (cPLA₂) or of a markerwhich is indicative of the presence or level of cPLA₂, wherein thepresence or level of said cPLA₂ or said marker is indicative of woundinfection, wherein the wound is an acute traumatic laceration.
 12. Amethod of diagnosing or prognosing wound infection, comprising testing awound fluid for the presence or level of high molecular weightphospholipase A₂ (cPLA₂) or of a marker which is indicative of thepresence or level of cPLA₂, wherein the presence or level of said cPLA₂or said marker is indicative of wound infection, wherein the wound is asurface-exposed wound.